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Newcastle Disease Virus (NDV) is a virus well known in the art [Diseases of poultry, 10th edition, edited by B. W. Calnek, Mosby International, Iowa State University Press, Ames, Iowa, 1997]. The virus is responsible for great economic losses in the poultry industry. It is also well known that many strains of NDV exist (EP 0770397 B1) with an enormous range in the severity and type of disease produced in poultry. Newcastle disease virus (NDV) is classified as an avian paramyxovirus-1 (APMV1), a member of the family Paramyxoviridae in the order Mononegavirales. Members of this family have a single stranded, linear, RNA, with an elliptical symmetry. The total genome is roughly 16,000 nucleotides. Replication of the virus takes place in the cytoplasm of the host cell.
This family is divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae. During 1993, the International Committee on the Taxonomy of Viruses rearranged the paramyxoviruses and placed NDV within the Rubulavirus genus. The genomes of most rubulaviruses, except NDV, contain a small hydrophobic (SH) protein gene that is not present among other paramyxoviruses. Based on predicted amino acid sequences for each viral protein, NDV clones phylogenetically group as a clade separate from the rubulaviruses. The polycistronic phosphoprotein (P) gene editing sequence of NDV and putative gene products are more similar to expression patterns among members of the Respirovirus and Morbillivirus. In addition, structure of the nucleocapsid protein more closely resembles the Respiroviruses. There are nine recognized serotypes among avian paramyxoviruses that infect primarily only bird species. These virus types are phylogenetically distinct from NDV, but separate as a clade with NDV from the other paramyxoviruses. This relationship was further confirmed by phylogenetic analysis of full-length genomic sequences.
As with the situation for many other avian viruses, NDV has evolved among birds separate from their mammalian counterparts. Consequently, based on several key factors, including gene and predicted amino acid sequences, avian paramyxoviruses deserve their own genus designation among the Paramyxovirinae.
Newcastle disease (ND) is a contagious viral disease affecting only species of birds. Clinical signs are extremely variable depending on the strain of virus, species and age of bird, concurrent disease, and pre-existing immunity.
Vaccination plays a pivotal role in the control of Newcastle disease (ND) in poultry. This can partly be attributed to the fact that several naturally derived less pathogenic and attenuated live viral strains were identified and have been available for this purpose as early as the second half of the 1940's (reviewed by Lancaster, 1964).
The extreme variation in virulence of different ND virus clones and the widespread use of live vaccines means that the identification of an clone as ND virus from birds showing clinical signs does not confirm a diagnosis of ND, so that an assessment of the virulence of the clone is also required.
NDV strains have been classified in several ways by several authors and institutions. An early form of classification was based on their pathogenicity, in which the strains were classified into velogenic, mesogenic, lentogenic and avirulent groups (Hanson and Brandly, 1955).
Several potential in-vitro tests for establishing virulence usually related to the molecular basis for pathogenicity are being investigated by various groups around the world. At present, a definitive assessment of virus virulence is usually based on one or more of the following in-vivo tests, although the current OIE definition allows molecular assessment of virulence.
1. The plaque size and the virulence relationship of the NDV strains was published by G. M. Schloer and R. P. Hanson (J. Virol. 1968 January; 2(1): 40-47.). Schloer and Hanson found that the size of the plaques of the NDV was related to virulence for chickens. Markedly larger plaques were produced by the velogenic (high virulence) strains while smaller plaques were found in mesogenic (intermediate virulence) strains. This method was used in the past as a way to classify NDV strains by measurement of plaque size.2. Mean death time in eggs The MDT has been used to classify ND virus strains into velogenic (taking under 60 hours to kill); mesogenic (taking between 60 and 90 hours to kill); and lentogenic (taking more than 90 hours to kill).3. Intracerebral pathogenicity index (ICPI). The most virulent viruses will give indices that approach the maximum score of 2.0, whereas lentogenic strains will give values close to 0.0. The mesogenic strains fall between 0.7 and 1.5.4. Intravenous pathogenicity index (IVPI). Lentogenic strains and some mesogenic strains will have IVPI values of 0, whereas the indices for virulent strains will approach 3.0.5. Molecular basis for pathogenicity. During replication, ND virus particles are produced with a precursor glycoprotein, F0, which has to be cleaved to F1 and F2 for the virus particles to be infectious. This post-translation cleavage is mediated by host-cell proteases. Trypsin is capable of cleaving F0 for all ND virus strains. It would appear that the F0 molecules of viruses virulent for chickens can be cleaved by a host protease or proteases found in a wide range of cells and tissues thus spreading throughout the host, damaging vital organs, but F0 molecules in viruses of low virulence are restricted in their sensitivity to host proteases resulting in restriction of these viruses to growth only in certain host-cell types. Most ND viruses that are pathogenic for chickens have the sequence 112R/K-R-Q-K/R-R116 of SEQ ID NO: 4 at the C-terminus of the F2 protein and F (phenylalanine) at residue 117, the N-terminus of the F1 protein, whereas the viruses of low virulence have sequences in the same region of 112G/E-K/R-Q-G/E-R116 of SEQ ID NO: 5 and L (leucine) at residue 117. It appears to be the requirement of at least one pair of basic amino acids at residues 116 and 115 plus a phenylalanine at residue 117 and a basic amino acid (R) at 113 if the virus is to show virulence for chickens. Based on these molecular findings the veterinary classification of ND viruses is no longer divided into three—but rather into two divisions—pathogenic and apathogenic.
It seems likely that the vast majority of birds are susceptible to infection with ND viruses of both high and low virulence for chickens, although the clinical signs seen in birds infected with ND virus vary widely and are dependent on factors such as: the virus, host species, age of host, infection with other organisms, environmental stress and immune status. In some circumstances infection with the extremely virulent viruses may result in sudden high mortality with comparatively few clinical signs. Thus the clinical signs are variable and influenced by other factors so that none can be regarded as pathognomonic.
Newcastle disease is defined as an infection of birds caused by a virus of avian paramyxovirus serotype 1 (APMV-1) that meets one of the following criteria for virulence:                A) The virus has an intracerebral pathogenicity index (ICPI) in day-old chicks (Gallus gallus) of 0.7 or greater        B) Multiple basic amino acids have been demonstrated in the virus (either directly or by deduction) at the C-terminus of the F2 protein and phenylalanine at residue 117, which is the N-terminus of the F1 protein. The term ‘multiple basic amino acids’ refers to at least three arginine or lysine residues between residues 113 and 116. Failure to demonstrate the characteristic pattern of amino acid residues as described above would require characterization of the cloned virus by an ICPI test. In this definition, amino acid residues are numbered from the N-terminus of the amino acid sequence deduced from the nucleotide sequence of the F0 gene, 113-116 corresponds to residues −4 to −1 from the cleavage site.’        
Genetic analyses of NDV strains cloned in the past 80 years have revealed the existence of at least 9 genotypes (and further subtypes) that showed not only region specific and host species associations but their temporal distribution was also apparent (Lomniczi és Czeglédi, 2005). It was shown that early genotypes [II.-IV. and Herts'33(W)] prevalent before the 1960s were replaced by recent genetic groups (V.-VIII.) following the introduction of vaccination. Recently sublineages of the Far East genotype VII have spread to other geographic areas, e.g. to Europe (see family tree of known NDV virus strains (FIG. 31)). Replacement of genotypes appears to be an evolutionary process rather than random epidemiological event in the distribution of NDV strains. The emergence of novel virulent genotypes seems to be inconsistent with the application of vaccination but experimental infections shed light on the process whereby immunized chicken population became the reservoir of the novel virulent viruses.
As to the ecology two major reservoirs of NDV strains exist in nature. The primordial reservoir consists of wild water-bird species that harbor primitive (apathogenic) viruses but, surprisingly, only two genetic lineages are known in the wild: class I and genotype I (belonging to class II). By contrast, the remainder (genotypes II.-VIII). comprises virulent strains and is maintained in the secondary (artificial) reservoirs of chickens. It is hypothesized that the chicken populations were seeded with apathogenic viruses and pathogenic strains emerged in the chicken host. Prior to the immunization period at least two independent colonizations could have taken place (with genotype I and II).
Genetic analysis of an authentic sample of the first European clone, Herts'33 (cloned in England in 1933), revealed that it represented a highly diverged novel early lineage. Contrarily to a 1940 publication from England in which the derivation of strain H, one of the most successful early vaccines, from Herts'33(W) by egg passage was reported, genetic analysis precluded relationships between them.
Genetic analyses of NDV strains have also indicated a remarkable genetic stability of NDV strains, even after prolonged and repeated passage. The genetic stability is proven by the lack of viral recombination in nature. Toyoda et al. analyzed the sequences of the NH and F genes of multiple strains of NDV cloned over a period of 50 years. There was no gene exchange by recombination in the generation of three lineages. (Toyoda T., Newcastle disease virus evolution. II. Lack of gene recombination in generating virulent and avirulent strains. Virology 169: 273-282, 1989.)
NDV is usually thought to be an avian virus, but it also able to infects humans. Although NDV causes a potentially fatal, noncancerous disease (Newcastle disease) in birds, it causes only minor illness, manifested in mild flu like symptoms, or conjunctivitis in exposed humans (historically chiefly observed in laboratory workers).
In 1971 a scientific publication in “The Lancet” by Dr. Laszlo Csatáry described case histories of cancer treatment with an undisclosed strain of Newcastle Disease Virus (The Lancet, 1971, 7728, p. 825). Subsequently to this publication Dr. L. Csatáry and co-workers have published a number of scientific publications as well as patent applications (see below) based on scientific work with a virus strain referred to as “MTH-68/H”. However, none of these references disclose the exact nature of the virus strain, the virus strain has never been commercially available nor been deposited at any virus library and therefore all these publications are not enabling for an expert skilled in the art. Moreover, further scientific work exists with virus compositions referred to as “MTH-68/H” from scientists other than Dr. L. Csatáry. However, it is highly unclear whether the virus compositions used are identical to that having been described in the Lancet publication cited above.
The work of Dr. Csatáry has obviously stimulated the medical community so that further work has been published such as EP 696326 B1 (Wellstat Biologics Corporation) and others as listed below. However, the scientific work disclosed therein cannot be reproduced by an expert in the art as the strains used therein such as strain 73-T or MK-107 are likewise not available to the public.
Significant interest has developed in the potential for use of NDV in cancer therapy because NDV has been found to have selective cell killing properties in many types of cancer cells while not effecting normal non-neoplastic cells. A report indicating that NDV might be useful as a cancer treatment was published in the early 1960s. Since then, a number of studies have been reported.
Many NDV strains have been found to be cytotoxic to cancer cells. Some strains are able to replicate in and destroy cancer cells while at the same time not effecting normal cells. These strains have been termed oncolytic. Different strains demonstrate different levels of cancer cell damaging properties, and different cancer cell types show sensitivity to different strain types of NDV. These properties define a strain's oncolytic potency. The oncolytic potency is thought to have a clinical correlation as to the therapeutic dosaging requirements. In experimental conditions the more oncolytically potent the strain is for a cancer cell type the lower the multiples of infective (MOI) viral particles per cell is needed to be able to observe a cytolytic effect. The clinical implication is a need for lower viral doses to achieve a therapeutic effect. While NDV has not been observed to cause any significant disease in humans—at extremely high therapeutic doses given parentally, as has been used in some clinical trials—it has been noted to potentially cause side effects of hypotension and high fever—leading to the need to find alternative techniques of dosaging—to desensitize the patient prior to giving the high dose application (see WO 00/62735).